Elda Grabocka

RAS oncogenes: weaving a tumorigenic web

RAS proteins are essential components of signalling pathways that emanate from cell surface receptors. Oncogenic activation of these proteins owing to missense mutations is frequently detected in several types of cancer. A wealth of biochemical and genetic studies indicates that RAS proteins control a complex molecular circuitry that consists of a wide array of interconnecting pathways. In this Review, we describe how RAS oncogenes exploit their extensive signalling reach to affect multiple cellular processes that drive tumorigenesis.

Human Pancreatic Cancer Tumors Are Nutrient Poor and Tumor Cells Actively Scavenge Extracellular Protein

Glucose and amino acids are key nutrients supporting cell growth. Amino acids are imported as monomers, but an alternative route induced by oncogenic KRAS involves uptake of extracellular proteins via macropinocytosis and subsequent lysosomal degradation of these proteins as a source of amino acids. In this study, we examined the metabolism of pancreatic ductal adenocarcinoma (PDAC), a poorly vascularized lethal KRAS-driven malignancy. Metabolomic comparisons of human PDAC and benign adjacent tissue revealed that tumor tissue was low in glucose, upper glycolytic intermediates, creatine phosphate, and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such accumulation could arise from extracellular proteins being degraded through macropinocytosis in quantities necessary to meet glutamine requirements, which in turn produces excess of most other amino acids. Consistent with this hypothesis, active macropinocytosis is observed in primary human PDAC specimens. Moreover, in the presence of physiologic albumin, we found that cultured murine PDAC cells grow indefinitely in media lacking single essential amino acids and replicate once in the absence of free amino acids. Growth under these conditions was characterized by simultaneous glutamine depletion and essential amino acid accumulation. Overall, our findings argue that the scavenging of extracellular proteins is an important mode of nutrient uptake in PDAC.

Wild-type H- and N-Ras promote mutant K-Ras driven tumorigenesis by modulating the DNA damage response

Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anticancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here, we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways and, consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo.

Disruption of oligomerization induces nucleocytoplasmic shuttling of leukemia-associated rho Guanine-nucleotide exchange factor.

The rgsRhoGEFs comprise a subfamily of three guanine nucleotide exchange factors, which function in linking heterotrimeric G-proteins to the monomeric RhoGTPase. Here, we reveal the novel finding that oligomerization of leukemia-associated RhoGEF (LARG) functions to prevent nucleocytoplasmic shuttling and to retain LARG in the cytoplasm. We establish that oligomerization is mediated by a predicted coiled-coil sequence (amino acids 1507–1520) in the extreme C terminus of LARG and that substitution of isoleucines 1507/1510 with alanines disrupts homo-oligomerization and leads to nucleocytoplasmic shuttling via the CRM1 nuclear transport pathway. In addition, we demonstrate that induced dimerization of an otherwise nuclear monomeric LARG mutant promotes cytoplasmic localization. Furthermore, we establish that nuclear import of monomeric LARG is mediated by the nuclear localization sequence 29PTDKKQK35 in the extreme N terminus. We propose that nucleocytoplasmic shuttling provides a mechanism for spatially regulating the activity of LARG toward its cytoplasmic targets and potentially new nuclear targets.

Functional consequences of Gα13 mutations that disrupt interaction with p115RhoGEF

The G-protein α subunit, α13, regulates cell growth and differentiation through the monomeric Rho GTPase. α13 activates Rho through direct stimulation of the guanine nucleotide exchange factor p115RhoGEF, which contains a regulator of G-protein signaling homology domain (RH) in its N-terminus. Through its RH domain, p115RhoGEF also functions as a GAP for Gα13. The mechanism for the Gα13/p115RhoGEF interaction is not well understood. Here, we determined specific α13 residues important for its interaction with p115RhoGEF. GST-pulldowns and co-immunoprecipitation assays revealed that individually mutating α13 residues Lys204, Glu229, or Arg232 to opposite charge residues disrupts the interaction of activated α13 with the RH domain of p115RhoGEF or full-length p115RhoGEF. We further demonstrate that mutation of Glu229, and to a lesser extent Lys204 or Arg232, disrupts the ability of activated α13 to induce the recruitment of p115RhoGEF to the plasma membrane (PM) and to activate Rho-mediated serum response element-luciferase gene transcription. Interestingly, an α13 mutant where a conserved Gly was mutated to a Ser (G205S) retained its ability to bind to p115RhoGEF, induce p115RhoGEF recruitment to the PM, and activate Rho-dependent signaling, even though identical Gly to Ser mutations in other α disrupt their interaction with regulator of G-protein signaling (RGS) proteins. These results demonstrate that, whereas several features of a typical α/RGS interaction are preserved in the α13/p115RhoGEF interaction, there are also significant differences.

Molecular Pathways: Targeting the Dependence of Mutant RAS Cancers on the DNA Damage Response

Of the genes mutated in cancer, RAS remains the most elusive to target. Recent technological advances and discoveries have greatly expanded our knowledge of the biology of oncogenic Ras and its role in cancer. As such, it has become apparent that a property that intimately accompanies RAS-driven tumorigenesis is the dependence of RAS-mutant cells on a number of nononcogenic signaling pathways. These dependencies arise as a means of adaptation to Ras-driven intracellular stresses and represent unique vulnerabilities of mutant RAS cancers. A number of studies have highlighted the dependence of mutant RAS cancers on the DNA damage response and identified the molecular pathways that mediate this process, including signaling from wild-type Ras isoforms, ATR/Chk1, and DNA damage repair pathways. Here, we review these findings, and we discuss the combinatorial use of DNA-damaging chemotherapy with blockade of wild-type H- and N-Ras signaling by farnesyltransferase inhibitors, Chk1 inhibitors, or small-molecule targeting DNA damage repair as potential strategies through which the dependence of RAS cancers on the DNA damage response can be harnessed for therapeutic intervention. Clin Cancer Res; 21(6); 1243–7. ©2014 AACR.

Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission

This study demonstrates a novel and unexpected role in cytokinesis for leukemia-associated RhoGEF (LARG). Depletion of LARG results in delayed abscission, and thus LARG is the first RhoGEF to be implicated in late cytokinesis.

Mitotic-dependent phosphorylation of leukemia-associated RhoGEF (LARG) by Cdk1

Rho GTPases are integral to the regulation of actin cytoskeleton-dependent processes, including mitosis. Rho and leukemia-associated Rho guanine-nucleotide exchange factor (LARG), also known as ARHGEF12, are involved in mitosis as well as diseases such as cancer and heart disease. Since LARG has a role in mitosis and diverse signaling functions beyond mitosis, it is important to understand the regulation of the protein through modifications such as phosphorylation. Here we report that LARG undergoes a mitotic-dependent and cyclin-dependent kinase 1 (Cdk1) inhibitor-sensitive phosphorylation. Additionally, LARG is phosphorylated at the onset of mitosis and dephosphorylated as cells exit mitosis, concomitant with Cdk1 activity. Furthermore, using an in vitro kinase assay, we show that LARG can be directly phosphorylated by Cdk1. Through expression of phosphonull mutants that contain non-phosphorylatable alanine mutations at potential Cdk1 S/TP sites, we demonstrate that LARG phosphorylation occurs in both termini. Using phosphospecific antibodies, we confirm that two sites, serine 190 and serine 1176, are phosphorylated during mitosis in a Cdk1-dependent manner. In addition, these phosphospecific antibodies show phosphorylated LARG at specific mitotic locations, namely the mitotic organizing centers and flanking the midbody. Lastly, RhoA activity assays reveal that phosphonull LARG is more active in cells than phosphomimetic LARG. Our data thus identifies LARG as a phosphoregulated RhoGEF during mitosis.

Raf-1 and Squamous Cell Carcinoma: Rok-ing the Boat

Squamous cell carcinoma (SCC) is the second most common form of nonmelanoma skin cancer. In this issue of Cancer Cell, Ehrenreiter et al. unveil a critical role for the Raf-1/Rok-alpha interaction in the pathogenesis of SCCs, thus paving the way for the development of therapeutic modalities to treat this malignancy.

Histological image processing features induce a quantitative characterization of chronic tumor hypoxia

Hypoxia in tumors signifies resistance to therapy. Despite a wealth of tumor histology data, including anti-pimonidazole staining, no current methods use these data to induce a quantitative characterization of chronic tumor hypoxia in time and space. We use image-processing algorithms to develop a set of candidate image features that can formulate just such a quantitative description of xenographed colorectal chronic tumor hypoxia. Two features in particular give low-variance measures of chronic hypoxia near a vessel: intensity sampling that extends radially away from approximated blood vessel centroids, and multithresholding to segment tumor tissue into normal, hypoxic, and necrotic regions. From these features we derive a spatiotemporal logical expression whose truth value depends on its predicate clauses that are grounded in this histological evidence. As an alternative to the spatiotemporal logical formulation, we also propose a way to formulate a linear regression function that uses all of the image features to learn what chronic hypoxia looks like, and then gives a quantitative similarity score once it is trained on a set of histology images.